We read with interest the recent article in this journal on screening for family members of nasopharyngeal carcinoma (NPC) patients by serologic tests against Epstein-Barr virus (EBV).1 Although several previous studies2, 3, 4, 5 have reported on the use of EBV serology for screening and early detection of NPC, the role of EBV DNA detection as a screening test for NPC remains to be defined. Circulating cell-free EBV DNA is detectable in the plasma of 96% of NPC patients6 by quantitative real-time polymerase chain reaction (PCR) and has been shown to be a useful marker in prognostication7 as well as residual disease detection.8 Studies on EBV DNA detection, however, are performed on known NPC patients but not in the general population. We report our preliminary results on plasma EBV DNA detection, in association with EBV serology, as screening tests for NPC in the setting of routine health assessment.
In the 6-month period from July to December 2004, a total of 1,475 blood samples were referred from the Health Assessment Centre of our hospital for detection of EBV status as part of body checkup. They comprised 875 male patients and 600 female patients with a median age of 49 years (range 16–92 years). None of them had prior history of EBV-related malignancies. All patients were tested for cell-free plasma EBV DNA level, and when the result was positive, serum samples were retrieved for EBV serologic testing.
The RealArt EBV LC PCR Kit (Artus, Hamburg, Germany), working on the LightCycler instrument (Roche Diagnostics, Mannheim, Germany), was employed for plasma EBV DNA detection. The EBV LC PCR Master reagent contained reagents and enzymes for the specific amplification of a 97 bp region of the EBNA1 gene of the EBV genome. Quantification standards were provided with the kit and were included in each run so as to generate the standard curve for EBV load determination. An external quantified EBV viral DNA control (Advanced Biotechnologies, Columbia, MD) was included in each run to check the performance of the assay. Serum EBV VCA IgA and EA IgA antibody levels were determined by indirect immunofluorescence assay on Raji cells and B95-8 cells induced to express EA and VCA, respectively (purchased from the Department of Microbiology, The University of Hong Kong). Antibody titer was defined as the highest dilution that yielded a positive fluorescent signal. Cutoff values for positivity were set at 1:40 for VCA IgA and 1:10 for EA IgA.
The presence of EBV DNA was detected in 33 patients (2.24%). Among them, 4 were positive for VCA IgA in which 1 patient was also positive for EA IgA. The clinical records of these patients were reviewed. The only patient with detectable EBV DNA (118 copies/mL) and positive VCA (titer 1:80) as well as EA (titer 1:40) IgA was a 48-year-old man who was referred for otorhinolaryngology (ENT) consultation. Endoscopic examination of the nasopharynx and biopsy showed undifferentiated carcinoma consistent with NPC. Another patient, a 20-year-old man who showed detectable EBV DNA (476 copies/mL) but negative EBV serology, exhibited bilateral enlarged tonsils and cervical lymphadenopathy suggestive of acute EBV infection, i.e., infectious mononucleosis, but defaulted follow-up. The other patients had no clinical evidence of acute EBV infection or EBV-related disorders. They were advised to monitor EBV status at regular intervals and consider ENT consultation if symptoms might arise.
Excluding the 2 aforementioned cases and a Caucasian patient, the median plasma EBV DNA level among 30 normal Chinese individuals (i.e., 2.03% of our cohort) was 31 copies/mL and mean ± SD of 104.6 ± 144.5 copies/mL, with a range of 1–532 copies/mL.
Previous reports from Hong Kong show that low copy numbers of plasma EBV DNA can be found in 3–7% of normal persons6, 9 as detected by quantitative real-time PCR. We confirm, with a much larger cohort than previous studies, the presence of detectable plasma EBV DNA in around 2% of apparently normal individuals in Hong Kong. The different EBV DNA targets, for example BamH1-W or EBNA1, may contribute to the difference in the detection rate. Since EBV virtually infects all humans by the time of adulthood, the presence of detectable levels of plasma EBV DNA most probably represents transient viral reactivation.10 Admittedly, plasma EBV DNA levels in most of our patients fall below the analytical sensitivity of the method so that results may not be entirely reproducible. Nevertheless, definite amplification signals are detectable in the test system that is adequately controlled. One patient in our cohort showed positivity for EBV DNA as well as VCA and EA IgA and was finally diagnosed as NPC.
Interpretation of our data, however, is limited by the fact that only a minority of patients with detectable EBV DNA undergo ENT examination and that currently no information is available on serial monitoring of EBV DNA status of our study patients. These notwithstanding, as there is insufficient evidence on the effectiveness of EBV screening by either plasma DNA or EBV serology alone or in combination in reducing the mortality and morbidity from NPC on a population basis, a randomized controlled trial to assess the efficacy of both EBV serology and DNA as screening tools for NPC may be warranted, especially in high-prevalence areas such as Southern China and Hong Kong.